Interpretive Summary: Carotenoids play an essential role in plants and are important in human nutrition and health. The significant roles of carotenoids invoke an inclination towards understanding the mechanism of carotenoid biosynthesis in plants. Chinese cabbage is one of the most renowned vegetables in Asia due to its exceptional nutritional value. Orange head Chinese cabbage accumulates significant amount of carotenoids in comparison with white Chinese cabbage, which gives an incentive to breed orange head Chinese cabbage for further improving nutritional quality. The orange head Chinese cabbage is controlled by a single recessive gene, Br-or. In this study, InDel and SSR molecular markers were used to construct a high resolution genetic map and define carotenoid isomerase (BrCRTISO) as the candidate gene for Br-or. This study provides the key step for unraveling the molecular mechanism controlling the orange head trait in Chinese cabbage.

Technical Abstract:
Orange head Chinese cabbage accumulates significant amounts of carotenoids with enhanced nutritional quality. To develop molecular markers for breeding of Chinese cabbage lines with high carotenoid content and to isolate the candidate gene underlying carotenoid synthesis, we performed fine mapping of the orange locus in a F2S4 mapping population. Genetic analysis revealed that the phenotype of the orange head trait was controlled by a single recessive gene, Br-or. The F2S4 mapping population consisting of 1724 individuals was developed from the cross between parent lines 11J16 and 11S39-2 by continuous selfing of a single heterozygous individual. Twenty-one tightly linked Simple Sequence Repeats (SSR) and insertion/deletion polymorphisms (InDels) markers were obtained. High resolution genetic mapping of these markers in the F2S4 mapping population placed Br-indel2 and Br-indel1 at genetic distance of 0.1 and 0.2 cM, respectively, on either side of the Br-or locus. Based on comparison of these two marker sequences with the fully sequenced Brassica rapa genome, the Br-or locus was delimited to a 16.7-kb genomic region. Three open reading frames (ORFs) were predicted in the target region. ORF1 encoded carotenoid isomerase that involves in the isomerization of carotenoids. ORF1 was found to be co-segregated with the Bo-or locus and was the most likely candidate gene for Br-or. The information obtained here will facilitate the breeding of nutrient enriched Chinese cabbage through marker-assisted selection (MAS) and provide a platform for gaining a better understanding of regulation of carotenoid biosynthesis in the plants.